Post-translational protein modification by tyrosine sulfation has a widespread occurrence among proteins of multicellular eukaryotic organisms. The enzyme responsible for protein tyrosine sulfation has been identified as the Golgi membrane-bound tyrosylprotein sulfotransferase (TPST). Studies by us and others have revealed the existence of multiple isoforms of TPST in human, mouse, and zebrafish. The discovery of distinct, yet homologous, TPSTs raises some fundamental questions concerning their functional relevance. Do they exhibit differential substrate specificity and act upon different subsets of target proteins? Are they expressed in the same or different cell types/tissues/organs? Do they share the same pattern of expression at different stages during embryonic development onto maturity? Do they function separately or can they complement each other within the context of physiology? To find answers to these unresolved issues, we will use the zebrafish as an experimental model. The proposed research is driven by the hypothesis that post-translational tyrosine sulfation is critical to the functioning of proteins involved in multiple physiological processes. The different TPSTs found in zebrafish and other vertebrates serve to sulfate different subsets of proteins in the same cells or in different, albeit overlapping, populations of cell types or tissues, and possibly at different developmental stages, for distinct functional purposes. We address this hypothesis in three interrelated specific aims: 1) To express and purify the three zebrafish TPSTs and systematically characterize their differential enzymatic properties and substrate specificity. A variety of biochemical and molecular biological techniques, as well as RNA interference strategy, will be employed. 2) To identify the proteins that are subjected to tyrosine sulfation in zebrafish embryos and larvae at different developmental stages, as well as in adult male and female zebrafish. A previously developed methodology in conjunction with MALDI-TOF analysis will be used. 3) To clarify the ontogeny and cell type/tissue/organ distribution of the three zebrafish TPSTs, as well as the physiological consequences of the knockdown of individual TPST genes. The distribution and expression, during embryonic development, of each of the three TPSTs will be examined using molecular biology and microscopy techniques. The morpholino antisense oligonucleotide approach will be taken to investigate the physiological consequences of the knockdown of individual TPST genes. The proposed studies are expected to provide fundamental information concerning the biochemical properties and functional relevance of TPSTs and tyrosine-sulfated proteins in zebrafish. Identification of novel characteristics of the zebrafish TPSTs or discovery of novel tyrosine-sulfated proteins appearing at different developmental stages or in different cell types/tissues/organs may serve as a starting point to investigate further the biochemical and physiological relevance of TPSTs and tyrosine-sulfated proteins in humans. PUBLIC HEALTH RELEVANCE: The proposed studies are designed to obtain fundamental information concerning the biochemical properties and functional relevance of tyrosylprotein sulfotransferases (TPSTs) and tyrosine-sulfated proteins in zebrafish. Identification of novel characteristics of the zebrafish TPSTs or discovery of novel tyrosine-sulfated proteins appearing at different developmental stages or in different cell types/tissues/organs may serve as a starting point to further investigate the biochemical and physiological relevance of TPSTs and tyrosine-sulfated proteins in humans. [unreadable] [unreadable] [unreadable]